ErbB2 is frequently detected in high-grade human DCIS (50-60%) and ErbB2 gene amplification has been found in 80-85% of the non-invasive, premalignant, comedo-type DCIS tumors. Intriguingly, ErbB2 is overexpressed in 25-30% of invasive breast cancers. It is not clear how some of the ErbB2 overexpressing DCIS develop into invasive ductal carcinoma (IDC). Acute activation of ErbB2 in MCF10A mammary epithelial cells (10A.B2 MECs) grown in a three dimensional (3D) culture that simulates in vivo conditions of acini formation in the mammary gland has led to the generation of multi-acinar structures lacking invasive properties that share structural properties associated with DCIS. Thus, activated ErbB2 induces the early transformation of MEC but is not sufficient to induce invasive behavior. It was postulated that additional genetic/molecular events or much higher levels of ErbB2 are needed for MECs to acquire invasive properties. However, the mechanisms by which activated ErbB2 induces early transformation of MECs and the events involved in their acquisition of invasive properties are not clear. Interestingly, we found that 14-3-3zeta, a protein that participates in many important cellular processes, is co-expressed at high levels with ErbB2 in approximately 30% of DCIS lesions of breast cancer patients and is associated with recurrence of invasive and metastatic diseases. Increasing 14-3-3zeta expression by retroviral infection in the 10A.B2 MECs led to invasive multi-acinar structures in 3D culture in contrast to the lack of invasion in the vector controls. On the other hand, blocking 14-3-3zeta expression in the 10A.B2 cells by siRNA inhibited their development into multi-acinar structures and blocking 14-3-3zeta function with a 14 -3-3zeta interfering mutant led to the inhibition of ErbB2-mediated transformation in NIH3T3 cells. Therefore, we hypothesize that 14-3-3zeta contributes to ErbB2-mediated mammary carcinogenesis and 14-3-3zeta high expression confers invasive potential in ErbB2-activated MECs that may facilitate the progression of ErbB2-overexpressing DCIS to IDC. In this proposal, we will investigate the role and the mechanisms of 14-3-3zeta in the transformation and acquisition of invasiveness of ErbB2 activated MECs (Aims 1 and 2) and confirm the role of 14-3-3zeta in the progression of ErbB2-overexpressing DCIS to IDC in patients (Aim 3). These comprehensive approaches will define the role of 14 -3-3zetain ErbB2-mediated mammary carcinogenesis and invasion, and ultimately in the progression of ErbB2-overexpressing DCIS to IDC. The study will bring new insights into early intervention, diagnosis, and treatment that may benefit the high-risk group of patients with ErbB2 and 14-3-3zeta overexpressing tumors.